The present invention relates to a method of stabilizing and preserving a useful protein, and a composition capable of stably holding the biological activity of a useful protein. Particularly the present invention relates to a method of stabilizing and preserving a useful protein, particularly, interferon of a mammal such as a dog or cat, and a composition capable of stably holding the activity thereof.
Proteins, particularly enzymes, useful proteins having biological activity, and the like can be mass-produced at low cost by gene recombination technology, and are thus used in various fields, particularly medicines, diagnostics, and foods. On the other hand, proteins are inactivated when the primary structure thereof is damaged due to degradation, and the function thereof greatly depends upon the higher-order structure. Therefore, proteins also have a problem in that the higher-order structure is easily, damaged due to various outside factors (temperature, changes over time, light, and pH) depending upon the types of proteins, thereby losing their function (biological activity). Thus, research is performed on a method of stabilizing the higher-order structure of a protein, and maintaining the biological activity thereof.
At present, a useful general method of stabilizing a protein comprises mixing it with another protein (gelatin, albumin, serum, collagen, or the like). Mixing with gelatin or serum permits preservation for a relatively long period of time, and there are many known drug products of enzymes and biologically active proteins produced by such a mixing method (Japanese Unexamined Patent Publication Nos. 2-264728, 2-49734, 54-80406, and 56-68607).
Stabilization of interferon (IFN) which is a biologically active substance having an immunomodulation action, and an antiviral action and which attracts attention in medical applications has been described. Japanese Unexamined Patent Publication Nos. 60-228422, 60-34919, 61-137828 and 60-260523 disclose a stabilization method comprising mixing with albumin or gelatin. Examples of known compounds having the protein stabilizing action other than proteins include saccharides, particularly monosaccharides, disaccharides, and polysaccharides such as dextran and hydroxyethyl starch (Japanese Unexamined Patent Publication Nos. 59-181223, 61-44826 and 60-155136 and Japanese Examined Patent Publication No. 6-51641), cyclodextrin, and polyhydric sugar alcohols (Japanese Unexamined Patent Publication No. 58-92691, and Japanese Examined Patent Publication No. 3-500882). There is also a method of making dominant a dimer of IFN-xcex3 having high activity using lactobionic acid (Japanese Examined Patent Publication No. 3-501604).
There are many reports of mixtures of gum arabic and proteins in which an aqueous gum arabic solution is used as a dispersing agent for a medicine (Japanese Unexamined Patent Publication No. 6-321803). However, there are also special report examples including: an example in which in production of an antibody, the amount of the antibody produced is increased by administering a mixture of a protein as an antigen and gum arabic than by administering a protein alone (Japanese Examined Patent Publication No. 58-23847), an example in which anti-cancer activity is increased by a combination of an anti-cancer drug and a significant amount of gum arabic (Japanese Examined Patent Publication No. 3-127740), and an example in which the efficiency of transport of a drug (polypeptide) to specified cells is increased by synthesizing a complex of the drug and gum arabic (the specification of U.S. Pat. No. 5,554,386). However, there is not known method of stabilizing a useful protein, and no useful protein composition stabilized by mixing with gum arabic.
When a protein is used as a medical additive, particularly an additive for an injection drug, the additive itself is a heterogeneous protein for living organisms, and thus possibly causes allergy depending upon the amount of the additive added. Further, for gelatin derived from cattle and known as a protein which can be added to medicines for various purposes, it is difficult to securely avoid mixing with a protein which causes Bovine Spongiform Encephalopathy. As described above, various problems are pointed out. Therefore, there is demand for a compound other than protein, which is atoxic to living organisms, and which has the action to stabilize a protein. Known examples of such compounds include saccharides, polyhydric alcohols, and the like. However, it is difficult to stably maintain a biologically active protein in a storage form such as an aqueous solution or a freeze-dried solid in a wide pH range, and thus a stabilizer having higher safety and, efficacy is required.
As a result of intensive research, the inventors found that the activity of a protein can be stably maintained by mixing a useful protein and an aqueous solution of gum arabic having the basic structure of arabic acid. It was also found that a useful protein composition obtained by freeze-drying the mixture solution maintains high biological activity, leading to the achievement of the present invention.